GLUT3 is induced during epithelial-mesenchymal
transition and promotes tumor cell proliferation in nonsmall cell lung cancer
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Citation
Masin, Mark, Jessica Vazquez, Simona Rossi, Svenja
Groeneveld, Natasha Samson, Petra C Schwalie, Bart
Deplancke, et al. “GLUT3 is induced during epithelialmesenchymal transition and promotes tumor cell proliferation in
non-small cell lung cancer.” Cancer & Metabolism 2, no. 1
(2014): 11.
As Published
http://dx.doi.org/10.1186/2049-3002-2-11
Publisher
BioMed Central Ltd
Version
Final published version
Accessed
Wed May 25 22:45:00 EDT 2016
Citable Link
http://hdl.handle.net/1721.1/89189
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Creative Commons Attribution
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http://creativecommons.org/licenses/by/4.0
Masin et al. Cancer & Metabolism 2014, 2:11
http://www.cancerandmetabolism.com/content/2/1/11
RESEARCH
Cancer &
Metabolism
Open Access
GLUT3 is induced during epithelial-mesenchymal
transition and promotes tumor cell proliferation
in non-small cell lung cancer
Mark Masin1, Jessica Vazquez1, Simona Rossi2, Svenja Groeneveld1, Natasha Samson1, Petra C Schwalie3,
Bart Deplancke3, Laura E Frawley4, Jérôme Gouttenoire5, Darius Moradpour5, Trudy G Oliver6 and Etienne Meylan1*
Abstract
Background: Alterations in glucose metabolism and epithelial-mesenchymal transition (EMT) constitute two important
characteristics of carcinoma progression toward invasive cancer. Despite an extensive characterization of each of them
separately, the links between EMT and glucose metabolism of tumor cells remain elusive. Here we show that the
neuronal glucose transporter GLUT3 contributes to glucose uptake and proliferation of lung tumor cells that have
undergone an EMT.
Results: Using a panel of human non-small cell lung cancer (NSCLC) cell lines, we demonstrate that GLUT3 is strongly
expressed in mesenchymal, but not epithelial cells, a finding corroborated in hepatoma cells. Furthermore, we identify
that ZEB1 binds to the GLUT3 gene to activate transcription. Importantly, inhibiting GLUT3 expression reduces glucose
import and the proliferation of mesenchymal lung tumor cells, whereas ectopic expression in epithelial cells sustains
proliferation in low glucose. Using a large microarray data collection of human NSCLCs, we determine that GLUT3
expression correlates with EMT markers and is prognostic of poor overall survival.
Conclusions: Altogether, our results reveal that GLUT3 is a transcriptional target of ZEB1 and that this glucose
transporter plays an important role in lung cancer, when tumor cells loose their epithelial characteristics to become
more invasive. Moreover, these findings emphasize the development of GLUT3 inhibitory drugs as a targeted therapy
for the treatment of patients with poorly differentiated tumors.
Keywords: Epithelial-mesenchymal transition, Glucose transporter, GLUT3, Non-small cell lung cancer, SLC2A3, ZEB1
Background
The reprogramming of energy metabolism constitutes
one of the hallmarks of cancer [1]. In order to build the
necessary biomass required for proliferation, tumor cells
increase their glucose consumption [2]. Glucose transporters of the GLUT (SLC2A) family are at the first step
of cellular glucose utilization, mediating glucose entry by
facilitative diffusion. The GLUT family is composed of
14 members that transport glucose or other substrates
in different tissues, with different efficiencies [3]. In various tumor types, an increased expression of GLUT1 has
been reported [4]. Furthermore, GLUT1 levels are higher
* Correspondence: etienne.meylan@epfl.ch
1
Swiss Institute for Experimental Cancer Research, School of Life Sciences,
Ecole Polytechnique Fédérale de Lausanne, Lausanne 1015, Switzerland
Full list of author information is available at the end of the article
in colorectal cancer cell lines harboring KRAS or BRAF
mutations compared with isogenic clones and confer cell
survival properties in low-glucose conditions [5]. Interestingly, in an oncogenic Kras(G12D)-dependent mouse
model of pancreatic ductal adenocarcinoma (PDAC),
oncogene withdrawal led to reduced Glut1 expression and
glucose uptake [6]. In contrast to GLUT1, little is known
about the regulation and function of another glucose
transporter, GLUT3, in cancer. GLUT3 was originally referred to as the neuronal GLUT [7]; with a high affinity
for glucose (KM approximately 1.5 mM) and the highest
calculated turnover number of all glucose transporters, it
ensures efficient glucose uptake by neurons of the central
nervous system. With the exception of neurons and a few
hematopoietic cell types, GLUT3 is lowly or not expressed
in most organs of healthy adults. However, pathological
© 2014 Masin et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Masin et al. Cancer & Metabolism 2014, 2:11
http://www.cancerandmetabolism.com/content/2/1/11
GLUT3 expression has been reported in gastric, testicular,
ovarian, and non-small cell lung cancer (NSCLC) [8,9].
Recently, GLUT3 was shown to be highly expressed in
glioblastoma and to promote the growth of brain tumor
initiating cells [10]. GLUT3 was also reported to be a transcriptional target of NF-κB and HMGA1, in mouse embryonic fibroblasts and human colorectal tumor cells,
respectively [11,12]. However, the regulation and contribution of GLUT3 to lung tumor progression remain
unknown.
The epithelial-mesenchymal transition (EMT) is a
process that occurs early in embryonic development,
notably during gastrulation, in which epithelial cells
undergo cytoskeletal changes and lose cell-cell contacts
to gain mesenchymal traits and become more motile
[13]. During carcinoma progression, pathological EMT
occurs to promote tumor cell invasion and metastasis.
One of the essential steps in the EMT process is the loss
of E-cadherin, an adherens junction protein that maintains cell-cell adhesion and epithelial tissue integrity.
Mechanistically, the EMT process can be triggered by
different transcription factors that include TWIST,
ZEB1, ZEB2, SNAIL, and SLUG [14]. In our study, we
discovered a strong association between the EMT program and the induction of the glucose transporter
GLUT3 in NSCLC and extended this observation to
tumor cells from another cancer type, hepatocellular carcinoma (HCC). We demonstrate that GLUT3 is a direct
transcriptional target of ZEB1. We further show that
GLUT3 expression contributes to proliferation of lung
tumor cells and is an independent prognostic factor of
poor overall survival in NSCLC.
Methods
Plasmid constructs
Human GLUT3, mouse Snail, and mouse Zeb1 cDNAs,
purchased from Thermo Scientific (Waltham, MA, USA;
MHS1010-7429646, MMM1013-7510291, and MMM101399828709, respectively), were amplified by polymerase
chain reaction (PCR) using forward 5′-CTCATCGATGC
CACCATGGGGACACAGAAGGT-3′ and reverse 5′-CT
CCCCGGGTTAGACATTGGTGGTGG-3′ (GLUT3), forward 5′-CTCATCGATGCCACCATGCCGCGCTCCTTC3′ and reverse 5′-CTCGAATTCTCAGCGAGGGCCT
CC-3′ (Snail), and forward 5′-CTCATCGATGCCACCAT
GGCGGATGGCCCC-3′ and reverse 5′-CTCGAATTCCT
AAGCTTCATTTGT-3′ (Zeb1) oligos. The PCR products
were digested with ClaI and XmaI (GLUT3) or ClaI and
EcoRI (Snail and Zeb1) and cloned into identically
digested pRDI292-CMV lentiviral vector (gift of D. Trono,
EPFL, Lausanne). Genomic DNA was used to amplify by
PCR (a) an approximately 1,000-bp region ending
close to the transcription start site of human SLC2A3
(gene encoding GLUT3), with the oligos forward 5′-
Page 2 of 14
CTCGAGCTCGAGACTAGCAGAAAGTG-3′ and reverse 5′-CTCCTCGAGCGACAAGCCCCCAGCCCCAC
CCCACCCCACCCCACCCCCCTGAAGCAA-3′, or (b)
a region containing the SLC2A3 intron 2 sequence, with
the oligos forward 5′-CTCGAGCTCACTGGGGTCAT
CAATGCTCC-3′ and reverse 5′-CTCCTCGAGGGTTG
GTGGAAGAACAGAC-3′. After SacI and XhoI digestion, the fragments were cloned into an identically
digested luciferase reporter plasmid containing a minimal
promoter (kindly provided by J. Huelsken, EPFL, Lausanne)
to generate prom.-LUC or int.2-LUC constructs, respectively. Deletion of the E-box-like motif CACCTC from the
intron 2 sequence was achieved by site-directed mutagenesis, using oligos forward 5′-CCACTCTTTATAGTGA
TGCACATCCTG-3′ and reverse 5′-CATCACTATAAA
GAGTGGGAGGAAGAAC-3′, combined with the oligos
indicated above in (b). shRNAs specific to GLUT3 were
either from Thermo Scientific (TRCN0000042880) or
designed using the pSICOLIGOMAKER 1.5 program
(created by A. Ventura, Memorial Sloan-Kettering Cancer Center, New York). In the latter case, forward 5′TGCAAGGATGTCACAAGAAATTCAAGAGATTTCT
TGTGACATCCTTGCTTTTTTC-3′ and reverse 5′-TC
GAGAAAAAAGCAAGGATGTCACAAGAAATCTCT
TGAATTTCTTGTGACATCCTTGCA-3′ oligos were
annealed and ligated into a pSicoR lentiviral vector.
The fidelity of all the PCR amplifications and oligo
syntheses was confirmed by sequencing. Control pLKO.1
was from Thermo Scientific.
Immunoprecipitation
Cells (8 × 107 per immunoprecipitation) were lysed in
NP-40 buffer (0.2% NP-40, 150 mM NaCl, 20 mM Tris
pH 8.0, 10 mM EDTA) containing a protease inhibitor
cocktail (complete, Roche, Basel, Switzerland) and 1 mM
Na3VO4 for 15 min on ice, followed by three quick steps
of freezing in liquid N2 and thawing at 37°C. Pre-clearing
was achieved using sepharose-6B (Sigma-Aldrich, St.
Louis, MO, USA) for 60 min at 4°C on a rotating wheel.
Immunoprecipitation was performed using a 1:1 mixture
of sepharose-6B and protein-G sepharose (Sigma-Aldrich),
together with 2 μg control or ZEB1 antibody, overnight at
4°C on a rotating wheel. After four steps of washing in
lysis buffer, sample buffer was added, and the samples
were boiled and loaded on a polyacrylamide gel for electrophoresis followed by Western blot.
Western blotting
Except when used for immunoprecipitation, cells were
lysed in RIPA buffer (20 mM Tris pH 8, 50 mM NaCl,
0.5% Na-deoxycholate, 0.1% SDS, 1 mM Na3VO4, protease inhibitor cocktail (complete, Roche)) for 5 min on
ice. Proteins were loaded on 8% or 10% polyacrylamide
Masin et al. Cancer & Metabolism 2014, 2:11
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gels for electrophoresis (150 V, 1 h). Transfer was performed on PVDF membranes (100 V, 1 h).
Cell culture conditions
The human embryonic kidney (HEK) 293 T cells and the
human hepatoma cell lines, which include HLF, HLE,
Huh-1 (all three kindly provided by K. Morikawa, Showa
University, Tokyo, Japan), Huh-7, and Hep3B, were
grown in DMEM. The human non-small cell lung cancer cell lines include A549, SW1573, NCI-H23, NCIH2122, NCI-H441, NCI-H460, NCI-H727, NCI-H2009,
NCI-H1944, and Calu-6; they were all obtained from
ATCC (Manassas, VA, USA) and all grown in RPMI. All
cell media were supplemented with 10% FBS. For the experiments with high or low glucose, cells were cultured
for 4 days in normal RPMI (containing 11 mM glucose),
supplemented by 10% FBS, or with RPMI without glucose, supplemented by 10% FBS, resulting in a final concentration of 11.5 mM and 0.5 mM glucose, respectively.
After 2 days of culture, the medium was replaced with
fresh medium to maintain the initial concentrations of
glucose.
Cell counting
To measure cell number, a trypan blue exclusion assay
was performed. Cells were mixed with trypan blue, and
the number of live, dead, and total cells was counted on
an automated cell counter (Countess, Life Technologies,
Carlsbad, CA, USA).
Page 3 of 14
which was then normalized to 1 to get fold activities.
For double transfection, 293 T cells were transfected first
with siRNAs and re-transfected 72 h later with plasmids,
24 h prior to lysis.
Anchorage-independent growth assays
Cells (9 × 104) were plated in triplicates in six-well
plates in 0.4% agar in RPMI on top of a layer of 0.8%
agar with RPMI. Cells were allowed to grow at 37°C
for 3 weeks. Colonies were counted using a microscope. A colony was defined as an aggregate of 50 or
more cells.
RNA purification, reverse transcription, and real-time PCR
amplification
RNA was purified using Trizol (Life Technologies), according to the manufacturer’s instructions. RNA (1 μg)
was reverse-transcribed using the High-Capacity cDNA
Reverse Transcription Kit (Life Technologies). cDNA
(5 ng) was used for real-time PCR amplification, using
commercially available Taqman probes for human 18S,
GAPDH, SLC2A3, SLC2A1, SLC2A4, SLC2A12, VIM,
ZEB1, SNAI1, and CDH1 (Life Technologies). Data were
normalized to GAPDH or 18S levels. For measurement of
mature miR-200b, reverse transcription was performed
with the TaqMan MicroRNA Reverse Transcription Kit
(Life Technologies), according to the manufacturer’s instructions, separately for miR-200b and the normalization
control, RNU24. cDNA (0.9 ng) was used for real-time
PCR amplification.
2-Deoxy-D-[3H]glucose uptake
Cells (1 × 106 per well, seeded the day before the experiment) were cultured on six-well plates. 2-Deoxyglucose
uptake assays were performed as described previously
[15]. Radioactivity was determined by scintillation counting. For sample normalization, protein concentration
was measured by BCA protein assay.
Transfection
siRNAs were from Life Technologies. siRNA transfection
was performed with RNAiMAX transfection reagent
(Life Technologies), according to the manufacturer’s
instructions. For each siRNA, 10 nM was used. cDNA
transfection of 293 T cells was performed with Lipofectamine 2000 (Life Technologies), according to the manufacturer’s instructions. For each well of a 12-well plate, a
mix of 125 ng Renilla-Luciferase (phRL-TK, Promega,
Madison, WI, USA) and 1.6 μg GLUT3-Luciferase (promoter, intron 2-WT or intron 2-Δ-CACCTC) was used.
Twenty-four hours after transfection, cells were lysed
and a luciferase reporter assay was performed (DualLuciferase Reporter Assay System, Promega). The reported luciferase activity was the ratio between Firefly
(GLUT3 construct)-Luciferase and Renilla-Luciferase,
Immunocytochemistry
Cells (4 × 107) were fixed in 4% paraformaldehyde, embedded in paraffin, and mounted on slides. Following
antigen retrieval with 10 mM Na-citrate and blocking,
cells were stained with anti-GLUT3 or anti-E-cadherin
antibodies overnight at 4°C, followed by washing and
staining with biotin-conjugated secondary antibodies for
1 h at room temperature. After washing, avidin-biotin
horseradish peroxidase complexes were added for 30 min
(ABC kit, Vectastain), and the complexes were revealed
with a DAB peroxidase substrate kit (Vector Laboratories,
Burlingame, CA, USA). Counterstain was performed using
Harris hematoxylin.
Reagents
Recombinant human TGF-β2 was from PeproTech
(Rocky Hill, NJ, USA). Antibodies used were anti-GLUT3
(#400062, Calbiochem, San Diego, CA, USA), anti-Ecadherin (#3195, Cell Signaling Technology, Danvers,
MA, USA), anti-vimentin (#5741, Cell Signaling), antiSNAIL (#3879, Cell Signaling), anti-ZEB1 (#sc-25388,
Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-βtubulin (#sc-9104, Santa Cruz Biotechnology), anti-CtBP
Masin et al. Cancer & Metabolism 2014, 2:11
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(#sc-17759, Santa Cruz Biotechnology), anti-p300 (#sc585, Santa Cruz Biotechnology), and normal rabbit IgG
(#12-370, Millipore, Billerica, MA, USA).
Virus production and infection
293 T cells were transfected using the calcium-phosphate
precipitation method, co-transfecting the lentiviral plasmid of interest in conjunction with pMD2G (VSV-G protein) and pCMVR8.74 (lentivirus packaging vector, kind
gift of D. Trono, EPFL, Lausanne). Viral supernatants
were harvested 24 and 36 h post-transfection, filtered,
and used directly for infection of cell lines, as described. Puromycin selection was performed to select
cells with stable pRDI292-CMV (delivering control, human GLUT3, mouse Snail, or mouse Zeb1 cDNA) or
pLKO.1 (with control or GLUT3 shRNA) genomic integration. Fluorescence-activated cell sorting was used to
select cells with stable pSicoR constructs, based on GFP
expression.
Statistics
Unless specified differently, P values were determined by
Student’s t tests.
ChIP-seq analysis
Publicly available replicate ChIP-seq data for ZEB1
(GSM803411), RNA polymerase II (GSM803485), H3K27ac
(GSM733771), as well as naked DNA (Input) (GSM733742)
[16] were re-analyzed using Bowtie 2 [17] for mapping
to the human GRCh37 genome with the parameters
‘–very-sensitive -M 10 -p 8’ and CCAT 3.0 [18] for
peak-calling using the parameters ‘fragmentSize 200 slidingWinSize 100 movingStep 50 isStrandSensitiveMode
1 minCount 13 outputNum 100000 randomSeed 123456
minScore 5 bootstrapPass 80’. Data were visualized using
IGV Browser [19].
ChIP-PCR
ChIP was performed as described previously [20], using
6 × 107 SW1573 and 5 μg of each antibody. To amplify
regions within the promoter region of SLC2A3, the following primers were used: 5′-ACTGCCCTGATAGTT
GGTCTGG-3′ with 5′-TTTGCCAGTGTTCCTTTCTT
CG-3′ (−608 and −523 bp upstream of SLC2A3 TSS); 5′ACTGCCCTGATAGTTGGTCTGG-3′ with 5′-GAGG
GAAAGACAGCCTGAGAGA-3′ (−608 and −482 bp
upstream of SLC2A3 TSS). To amplify regions from the
intron 2, the following primers were used: 5′-CATCA
CAGTTGCTACAATCGGC-3′ with 5′-ACCATGCCTG
GCCTTAAATTCT-3′ (2,396 and 2,559 bp downstream
of SLC2A3 TSS); 5′-ACCATGCCTGGCCTTAAATT
CT-3′ with 5′-AGCCTCAGGAGTAGCTGGGACT-3′
(2,452 and 2,594 bp downstream of SLC2A3 TSS); 5′GTGAGTGCCAGGCCACAATAAT-3′ with 5′-TGTGT
Page 4 of 14
TGCTCAGGATGGTGTTT-3′ (2,466 and 2,647 bp
downstream of SLC2A3 TSS); 5′-AGTCCCAGCTAC
TCCTGAGGCT-3′ with 5′-TTCCGGGAGTAAGTG
AGCTTTG-3′ (2,573 and 2,791 bp downstream of
SLC2A3 TSS); 5′-AAACACCATCCTGAGCAACACA3′ with 5′-TGAGGTGAAAGAGTGGGAGGAA-3′ (2,626
and 2,831 bp downstream of SLC2A3 TSS); 5′-TTCCT
CCCACTCTTTCACCTCA-3′ with 5′-GCACCGATGTT
CACAGTCTACC-3′ (2,810 and 2,926 bp downstream of
SLC2A3 TSS); 5′-AAGCTGGGTTCCCTTAGCAGAG-3′
with 5′-AAAGGGTTGGTGGAAGAACAGA-3′ (2,877
and 3,117 bp downstream of SLC2A3 TSS); 5′-CAGT
CTGTTCTTCCACCAACCC-3′ with 5′-AGGACCAGA
GAGACGTGAGCAG-3′ (3,093 and 3,212 bp downstream of SLC2A3 TSS). The intergenic region upstream
of GAPDH was amplified, using primers 5′-ATGGGTGC
CACTGGGGATCT-3′ and 5′-TGCCAAAGCCTAGGG
GAAGA-3′, as described previously [21]. As positive control, a DNA sequence of CDH1 known to bind ZEB1 was
amplified using primers 5′-GGCCGGCAGGTGAACCC
TCA-3′ and 5′-GGGCTGGAGTCTGAACTGA-3′, as
described previously [22]. Real-time PCR was performed
using SyBR green. Fold enrichment was calculated as
follows: 2^ΔΔct (ΔΔct = Δctspecific antibody − ΔctIgG). Data
were normalized using the negative control, an untranscribed region upstream of GAPDH, and represented
as fold enrichment/fold enrichment of the negative control locus.
Microarray analysis
We collected publicly available datasets from journal articles and Gene Expression Omnibus (GEO) repository,
selecting those with medium to large sample size [23,24].
The .CEL files were imported into R/Bioconductor (http://
www.bioconductor.org/) and RMA normalized [25]. A
total of 462 early-stage untreated lung NSCLC samples
were available, with associated patients’ overall survival.
The probe IDs with higher variation for each gene were
retained from a total of 13,960 genes. The normalized data
together with the clinico-pathological variables were then
used for further analysis. Pairwise Spearman correlations
between GLUT3 and other available genes were calculated. Univariate and multivariate survival analyses were
performed using proportional Cox regression (package
‘survival’, R). Kaplan-Meier figure was reported showing
hazard ratio (HR), 95% confidence intervals, and Wald
p_value from Cox regression model for a specific comparison of interest. The continuous GLUT3 expression
was dichotomized in high and low risk levels by the
median of the expression for visualization purposes.
Multivariate analysis was performed with the following
variables: GLUT3 continuous expression, stage, histology, gender, and age (dichotomized by the median of
62 years).
Masin et al. Cancer & Metabolism 2014, 2:11
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Page 5 of 14
Results
GLUT3 expression correlates with EMT
To determine if the neuronal glucose transporter GLUT3
is expressed in lung tumor cells, we used a panel of ten
human NSCLC cell lines. We noticed that GLUT3 expression, as determined by real-time PCR analysis, varied considerably between the cell lines, with five of them
expressing high and the other five expressing low GLUT3
mRNA levels (Figure 1A). Several molecular markers can
be used to classify human lung tumor cells according to
their EMT status [26]. We monitored the epithelial or
mesenchymal status in these cells by assessment of RNA
levels of E-cadherin and the mature form of miR-200b
B
2.5x104
GLUT3
E-cadherin
SW1573
1.5x104
1x104
H460
5x103
0
1.2x106
vimentin
mesenchymal
2x10
GLUT3
4
H23
1x106
H727
6x105
4x105
2x105
0
4x105
3.5x105
3x105
2.5x105
2x105
1.5x105
1x105
5x104
0
4500
4000
3500
3000
2500
2000
1500
1000
500
0
epithelial
8x105
H2009
E-cadherin
miR-200b
SW1573
H23
H460
A549
Calu-6
H441
H727
H1944
H2009
H2122
microRNA, rel. expression
mRNA, rel. expression
mRNA, rel. expression
mRNA, rel. expression
A
(two markers of the epithelial state), and vimentin (a
prototypical mesenchymal marker) using real-time PCR.
These three markers clearly separated the cell lines into
two groups: an ‘epithelial group’ (composed of H441,
H727, H1944, H2009, and H2122 cells) that expresses Ecadherin and miR-200b, but almost no vimentin, and a
‘mesenchymal group’ (SW1573, H23, H460, A549, and
Calu-6) that expresses high levels of vimentin, but very little E-cadherin and miR-200b. GLUT3 expression was elevated specifically in the mesenchymal group, whereas
levels were very low in cell lines of the epithelial group
(Figure 1A), strongly suggesting that this glucose transporter is up-regulated during or after an EMT. To
mesenchymal
epithelial
Figure 1 GLUT3 is strongly expressed in mesenchymal lung tumor cells. (A) The indicated cell lines were lysed for RNA preparation
followed by reverse transcription. The cDNA was amplified by real-time PCR using probes specific for the indicated genes or internal controls.
Data show means ± s.d. (n = 3) of mRNA or mature microRNA expression relative to the cell line expressing the least amount of the same gene
(set to 1). (B) The indicated cells were stained to analyze the expression of GLUT3 or E-cadherin by immunocytochemistry. Scale bar, 100 μm. In
(A) and (B), the dashed line indicates the separation between the mesenchymal and the epithelial groups of cells.
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Page 6 of 14
500
20
400
15
300
10
200
5
100
0
1800
1600
1400
1200
1000
800
600
400
200
0
7
48 h
72 h
96 h
0
25
C
H727
H2009
WB: SNAIL
29 175 ZEB1
SNAIL
ctl
ZEB1
SNAIL
WB: ZEB1
20
15
10
5
0
5
6
4
5
4
3
3
2
E-cadherin
24 h
mRNA, rel. expression
20
GLUT3
600
25
2
1
1
0
0
ZEB1
40
ctl
30
ctl
60
H2009
700
mRNA, rel. expression
80
0
H727
35
vimentin
B
vimentin
SNAIL
GLUT3
100
mRNA, rel. expression
ratio TGF-β treated/non treated
A
ZEB1
To test the hypothesis that GLUT3 is induced upon
EMT, we first stimulated the H2122 epithelial cell line
with TGF-β, a potent EMT-inducing factor. As expected,
ctl
GLUT3 is induced during EMT
TGF-β treatment up-regulated the expression of the
mesenchymal marker vimentin. More importantly, GLUT3
was concomitantly up-regulated in a temporal manner
(Figure 2A). Because TGF-β is pleiotropic [27], we then
directly tested the role of two known EMT-inducing transcription factors, SNAIL and ZEB1, in GLUT3 regulation.
We transduced two low-GLUT3 expressing epithelial cell
lines, H727 and H2009, with lentiviruses to generate cells
stably expressing either mouse SNAIL or ZEB1 protein.
As anticipated, each of H727-SNAIL, H727-ZEB1, H2009SNAIL, and H2009-ZEB1 stable cell populations showed a
downregulation of E-cadherin and an up-regulation of
vimentin. Furthermore, there was a 5- to 27-fold, and a
132- to 354-fold up-regulation of GLUT3 in H727 and
H2009 stable cells, respectively, compared to cells expressing a control plasmid (Figure 2B). Interestingly, and in
agreement with previous findings [28], stable expression
of SNAIL led to induction of endogenous ZEB1 (the reciprocal did not occur), raising the possibility that mouse
SNAIL induces GLUT3 expression indirectly through
ZEB1 (Figure 2C). Moreover, the extent of ZEB1 induction
correlated to that of GLUT3: in H727 cells, mouse SNAIL
SNAIL
determine if the correlation with EMT was specific for
GLUT3, or if other transporters within this family had a
similar expression pattern, we monitored the expression
of additional GLUT family members that carry glucose
(GLUT1, GLUT2, GLUT4, GLUT12, and GLUT14).
Whereas GLUT2 and GLUT14 were undetectable across
the cell lines (MM and EM, unpublished observations),
GLUT1, GLUT4, and GLUT12 were expressed, but none
of them showed any correlation with EMT (Additional
file 1). Next, to confirm that high GLUT3 mRNA expression in mesenchymal cells reflected actual protein
expression, we performed immunocytochemistry analyses.
This demonstrated that GLUT3 protein was expressed
and localized to the plasma membrane of mesenchymal
cells, but was not detectable in the cells from the epithelial
group (Figure 1B); once again, there was an inverse correlation between GLUT3 and E-cadherin expression.
Figure 2 GLUT3 is induced upon EMT. (A) H2122 cells were stimulated with 10 ng/ml TGF-β for the indicated time points, after which the cells
were lysed for RNA preparation followed by reverse transcription. The cDNA was amplified by real-time PCR using probes specific for the indicated
genes or internal controls. Data show means ± s.d. (n = 4) of mRNA expression ratios between TGF-β treated and non-treated conditions. (B) H727 or
H2009 cell populations stably expressing a control plasmid (ctl), SNAIL, or ZEB1 were lysed for RNA preparation followed by reverse transcription. The
cDNA was amplified by real-time PCR using probes specific for the indicated genes or internal controls. Data show means ± s.d. (n = 3) of mRNA
expression relative to the cell line expressing the least amount of the same gene (set to 1). (C) H727 or H2009 stable cell populations were lysed with
RIPA buffer to prepare protein extracts and to analyze the expression of the indicated proteins by Western blot.
Masin et al. Cancer & Metabolism 2014, 2:11
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overexpression resulted in a 2.5- and 27-fold induction of
human ZEB1 and GLUT3 mRNA, respectively, whereas it
was 9- and 354-fold in H2009 cells (Additional file 2).
Next, to assess if the link between GLUT3 and EMT is
specific for NSCLC, or whether it occurs in other tumor
types, we analyzed GLUT3 expression in human cell lines
derived from HCC. Based on marked differences in the
expression of vimentin and E-cadherin, we could distinguish two groups of cells: an epithelial group (Huh-7,
Hep3B, and Huh-1) and a mesenchymal group (HLF and
HLE). Similar to NSCLC cell lines, GLUT3 but not
GLUT1 expression was specifically elevated in the mesenchymal group, and very low in the epithelial group
(Figure 3A). Additionally, TGF-β treatment of the lowGLUT3 expressing cell line, Hep3B, led to increased expression of GLUT3, whereas GLUT1 levels remained
unchanged (Figure 3B). Altogether, these data demonstrate that the regulation of GLUT3 during EMT occurs
in at least two different cancer types, NSCLC and HCC.
ZEB1 induces GLUT3 through direct binding and
transcriptional activation
Although ZEB1 is best characterized as a transcriptional
repressor, depending on co-factor recruitment—and
probably other parameters—it can activate transcription.
For example, ZEB1 represses gene transcription when
bound to the co-repressor C-terminal-binding protein
(CtBP), whereas it activates target gene expression
when bound to p300, P300/CBP-associated factor
(P/CAF), or receptor-regulated (R)-SMADs [29-32].
Co-immunoprecipitation experiments performed on extracts from mesenchymal lung tumor cells revealed endogenous interactions between ZEB1 and CtBP, as well as
ZEB1 and p300, suggesting ZEB1 forms different complexes to trigger target gene repression or activation in
these cells (Additional file 3). To test the hypothesis that
ZEB1 directly activates GLUT3 (SLC2A3) gene transcription, ZEB1 mRNA and protein expression was first monitored in the panel of lung tumor cells. This showed that
ZEB1 was more strongly expressed in GLUT3-proficient,
mesenchymal cells compared to cells from the epithelial
group (Figure 4A and Additional file 1). Additionally, in
response to TGF-β stimulation (see Figure 2A), there was
an up-regulation of ZEB1, but not SNAIL, which preceded
that of GLUT3 (Additional file 4), suggesting ZEB1 induction enables the activation of GLUT3 transcription. Next,
we analyzed public data from lymphoblastoid cell extracts
subjected to chromatin immunoprecipitation using antiZEB1, anti-RNA polymerase II or anti-histone H3 (acetyl
K27) antibodies, followed by DNA sequencing (ChIP-seq)
[16]. SLC2A3 was actively transcribed in these cells, as inferred from the occupancy of RNA polymerase II and the
acetylation of histone H3 on Lysine 27 at the SLC2A3
locus (Figure 4B). Importantly, an interaction between
Page 7 of 14
ZEB1 and a region located within the second intron of
SLC2A3 was uncovered, with a peak of interaction that localized to an E-box-like motif, composed of a CACCTC
sequence (Figure 4B). Of note, ZEB1 has been reported to
bind such DNA sequences to regulate transcription
[33,34]. To determine if a similar ZEB1-SLC2A3 gene
interaction occurred in GLUT3-expressing lung tumor
cells, we performed ChIP-PCR analyses in extracts of
SW1573 cells. As positive and negative controls, respectively, we amplified a sequence of E-cadherin (CDH1)
known to bind ZEB1 and an intergenic sequence upstream of GAPDH [21,22]. ZEB1 bound to DNA sequences located within intron 2 of SLC2A3, but not to
sequences from the promoter region (Figure 4C). Having
established the localization of ZEB1 binding to the
SLC2A3 gene, we cloned the DNA sequence of intron 2
or, for comparison, a 1-kb sequence preceding the
SLC2A3 transcription start site into a luciferase reporter
plasmid (Figure 4D). Upon transfection into 293 T cells,
which express high amounts of endogenous ZEB1 (see
Figure 4A), the basal luciferase activity was significantly
stronger from the intron 2 construct than from the promoter construct. Also, luciferase activity from the intron 2
construct significantly decreased upon ZEB1 knockdown
(Figure 4D). Furthermore, deletion of the E-box-like motif
in intron 2 substantially reduced luciferase activity, revealing the CACCTC sequence as an important functional
element in response to ZEB1 (Figure 4E). Collectively,
these results demonstrate that GLUT3 is induced during
EMT by a mechanism involving direct binding and transcriptional activation by ZEB1.
GLUT3 promotes glucose uptake and proliferation of
mesenchymal lung tumor cells
We next sought to determine the functional consequences of high GLUT3 expression in mesenchymal
lung tumor cells using GLUT3 knockdown. Transfection
of small interfering (si)RNAs to GLUT3 into SW1573
cells caused a reduction in the number of live cells,
whereas GLUT1 knockdown did not or only slightly reduced cell number (Figure 5A and Additional file 5).
The proportion of dead cells was not changed upon
GLUT3 knockdown in SW1573 cells (Additional file 5),
suggesting that GLUT3 primarily regulates proliferation
of these cells, not survival. In contrast, H727, a lowGLUT3 expressor, was not affected by GLUT3 knockdown. Next, we transduced SW1573 cells with lentiviruses
delivering short hairpin (sh)RNAs targeting GLUT3 to
generate cells with stable GLUT3 knockdown (Additional
file 5). We first used these cells to interrogate the contribution of GLUT3 to glucose import. Knockdown of
GLUT3 with two independent shRNAs led to a decrease
of more than 50% in the efficiency of radioactive 2deoxyglucose incorporation, revealing that GLUT3 plays a
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B
6x103
5x103
4x103
3x103
2x103
1x103
0
GLUT1
35
30
25
20
15
10
5
0
TGF-β:
120
mRNA, rel. expression
GLUT3
GLUT3
7x103
mRNA, rel. expression
mRNA, rel. expression
8x103
-
+
vimentin
100
80
60
40
20
0
E-cadherin
6x103
5x103
4x103
3x103
2x103
1x103
N.D.
mRNA, rel. expression
7x103
mesenchymal
Huh-1
Huh-7
Hep3B
HLE
GLUT1
HLF
mRNA, rel. expression
0
20
18
16
14
12
10
8
6
4
2
0
epithelial
Figure 3 GLUT3 is strongly expressed in mesenchymal liver tumor cells. (A) The indicated cell lines were lysed for RNA preparation
followed by reverse transcription. The cDNA was amplified by real-time PCR using probes specific for the indicated genes or internal controls.
Data show means ± s.d. (n = 3) of mRNA expression relative to the cell line expressing the least amount of the same gene (set to 1). N.D., not
detected. (B) Hep3B cells were stimulated with 10 ng/ml TGF-β for 72 h, after which the cells were lysed for RNA preparation followed by reverse
transcription. The cDNA was amplified by real-time PCR using probes specific for the indicated genes or internal controls. Data show means ± s.d.
(n = 3) of mRNA expression relative to the untreated conditions (set to 1).
prominent role in glucose uptake of mesenchymal cells
(Figure 5B). Importantly, GLUT3 knockdown also led to
a reduction in the number of colonies grown in
anchorage-independent conditions (Figure 5C). Next, to
determine if GLUT3 knockdown had an impact on the
mesenchymal state of tumor cells, we monitored vimentin and E-cadherin expression. Interestingly, vimentin
expression was substantially decreased upon GLUT3
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Page 9 of 14
lung tumor cells
mesenchymal
WB:
293T
H2122
H2009
H727
H1944
H441
A549
Calu-6
H23
46 SW1573
β-tubulin
80 58 H460
ZEB1
epithelial
175 -
B
33 kb
8,070 kb
8,080 kb
8,090 kb
[0 - 20]
Input
[0 - 30]
H3K27ac
[0 - 60]
RNAPolII
[0 - 20]
ZEB1
ZEB1 peaks
CACCTC (-)
SLC2A3
C
Ex.1
D
Ex.2 Ex.3
Ex.1
Ex.2 Ex.3
SLC2A3
-1000
+1
SLC2A3
-1000
+2466 +3127
4
3
2
7
6
P=0.01 P=0.05
WB:
5
175 80 58 46 -
4
3
2
ZEB1
β-tubulin
LUC
int.2
WT
rel. luciferase activity
si ctl
si ZEB1
si ctl
CDH1
GAPDH
3093 - 3212
2877 - 3117
LUC
Δ-CACCTC
8
P=0.002
7
6
P=0.03
5
4
3
2
1
0
Figure 4 (See legend on next page.)
2810 - 2926
2626 - 2831
2573 - 2791
2466 - 2647
-608 - -482
-608 - -523
prom.
2452 - 2594
0
si ctl
1
1
si ZEB1
rel. luciferase activity
5
0
E
LUC
8
6
si ZEB1
7
int.2
LUC
INTRON 2
2396 - 2559
DNA binding (fold enrichment)
prom.
PROM.
Masin et al. Cancer & Metabolism 2014, 2:11
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Page 10 of 14
(See figure on previous page.)
Figure 4 GLUT3 is a direct ZEB1-target gene. (A) The indicated cell lines were lysed with RIPA buffer to prepare protein extracts and to
analyze the expression of ZEB1, or β-tubulin as control, by Western blot. (B) IGV Browser view of 33 kb around the SLC2A3 gene including ZEB1
(black), RNA Polymerase II (RNAPolII, green), the active histone mark H3K27ac (green-yellow) ChIP-seq, and Input (grey) tracks in human lymphoblastoid
cell lines. ZEB1 peak calls and ‘CACCTC’ sequence matches on the reverse strand are also indicated (black). The orientation of SLC2A3 is indicated by an
arrow. (C) Extracts from SW1573 cells were used for ZEB1 ChIP followed by real-time PCR. The coordinates of the PCR amplicons relative to the TSS are
indicated. CDH1 and an untranscribed sequence upstream of GAPDH were used as positive and negative controls for ZEB1 binding, respectively. The
data shown are representative of three independent experiments. (D) (Upper part) scheme showing the promoter region of 1,000 bp preceding the
transcription start site (prom.) and the intron 2 region (int.2), which were amplified from the SLC2A3 gene and cloned into the luciferase reporter
construct. Ex. exon, LUC luciferase. (Lower part) 293 T cells were transfected with the indicated luciferase reporter plasmids. Luciferase activity was
analyzed 24 h later. Data show means ± s.d. (n = 5) of relative luciferase activity. (Lower right) 293 T cells were transfected with control (ctl) or ZEB1
siRNAs and were lysed 96 h later to prepare protein extracts and to analyze the expression of ZEB1, or β-tubulin as control, by Western blot. (E) 293 T
cells were transfected with the indicated luciferase reporter plasmids. For the int.2 construct, either wild-type (WT) or a variant with a deletion of the
E-box-like motif (Δ-CACCTC) was used. Luciferase activity was analyzed 24 h later. Data show means ± s.d. (n = 5) of relative luciferase activity.
knockdown (Additional file 6). Hence, although we did
not observe any statistically significant variation in E-cadherin mRNA levels, these data suggest that GLUT3dependent glucose uptake participates in the maintenance
of a mesenchymal state. Finally, we wanted to determine if
increased GLUT3 expression is sufficient to impact on the
growth of lung tumor cells with epithelial traits. To do
this, we generated H727 cells stably expressing human
GLUT3 (Additional file 7). In high glucose concentrations,
there was no difference in the proliferation of cells overexpressing GLUT3 and control cells (Additional file 7).
However, we reasoned that because parental H727 cells
have adapted to grow in high glucose levels without
GLUT3, ectopic expression of this high-affinity glucose
transporter might affect proliferation specifically when
glucose concentrations are low. Indeed, the growth rate of
parental cells was diminished by a reduction in glucose
levels. Under these conditions, H727-GLUT3 cells grew
faster than control cells, demonstrating that GLUT3 expression is sufficient to increase cancer cell proliferation
in conditions where glucose concentrations are limiting
(Figure 5D). Altogether, these results demonstrate an important contribution for GLUT3 in lung tumor cell
proliferation.
GLUT3 expression in human NSCLC correlates with poor
overall survival and EMT signatures
To assess the relevance of our findings in vivo, we used
a combination of five publicly available microarray datasets of human NSCLC. This yielded 462 samples with
follow-up of overall survival. The analysis revealed that
high GLUT3 expression was associated with a poor overall survival (Figure 6A), a finding consistent with previous studies [10,35]. Additionally, a multivariate analysis
provided evidence for high GLUT3 expression as an independent predictor of poor overall survival (Table 1).
Next, we made pairwise comparisons between each of
SLC2A3, CDH1, and various EMT markers or inducers
(VIM, SNAI1, SNAI2, ZEB1, ZEB2, and TWIST1). As
expected, most mesenchymal genes had a positive
correlation with each other and a negative correlation
with CDH1. Importantly, all mesenchymal genes showed
a statistically significant positive correlation with
SLC2A3 expression (P < 0.001) (Figure 6B). SLC2A3 expression was also negatively correlated with CDH1, as
expected, although these data were not significant
(Figure 6B). Altogether, these in vivo findings support the
in vitro data that position GLUT3 as an important factor
in the process of EMT and lung tumor progression.
Discussion
In this study, we have identified a unique role for glucose transporter GLUT3 in the proliferation of lung
tumor cells with mesenchymal characteristics. We found
that GLUT3 is strongly up-regulated during EMT and contributes to glucose uptake specifically in mesenchymal-like
lung tumor cells. Furthermore, GLUT3 overexpression is
sufficient to substantially enhance the proliferation of lung
tumor cells with epithelial traits specifically in the context
of low glucose. Interestingly, increased glucose consumption was shown to promote EMT via stabilization of
SNAIL [36], suggesting that increased expression of
GLUT family members promotes the induction and/or
maintenance of EMT. In our experiments, we did not detect an EMT occurring in response to ectopic GLUT3
protein expression in epithelial cells (MM and EM, unpublished observations), but we found a decrease in
vimentin expression upon GLUT3 knockdown in mesenchymal cells. Collectively, these data suggest the intriguing
possibility that GLUT3 plays a role in the successful establishment or maintenance of the EMT, when tumor cells
become more motile and invasive, or in tumor cell survival upon loss of cell-cell adhesion.
EMT in cancer is a complex phenomenon that is orchestrated by different transcription factors with partially
overlapping gene targets. These transcription factors,
like SNAIL or ZEB1, often act as transcriptional repressors. However, the mechanisms of target gene regulation
are not fully elucidated. For example, ZEB1 inhibits the
transcription of target genes when bound to CtBP
Masin et al. Cancer & Metabolism 2014, 2:11
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Page 11 of 14
SW1573
3x106
1.5x106
P=0.02
P=0.0008
P=0.0006
2x106
P=0.0007
3x106
2.5x106
2x106
1.5x106
1x106
1x106
5x105
5x105
ctl
2
3
GLUT3
P=0.002
siRNA:
P=0.003
0.3
1
number of colonies
0.25
0.2
0.15
0.1
0.05
2
3
GLUT3
1
2
GLUT1
700 P=0.001
600
P=0.03
500
400
300
200
pSicoR
pLKO.1
pSicoR
GLUT3
ctl
0
shRNA:
GLUT3
GLUT3
ctl
ctl
shRNA:
GLUT3
100
0
D
1
2
GLUT1
C
0.35
glucose uptake
(nmol 2-DOG / mg prot x 10 min)
1
ctl
0
0
siRNA:
ctl
number of live cells
2.5x106
B
H727
3.5x106
pLKO.1
2x106
1.8x106
number of live cells
1.6x106
P=0.02
1.4x106
P=0.03
1.2x106
1x106
8x105
6x105
4x105
2x105
[glc]: high
ctl
GLUT3
parental
parental
0
low
Figure 5 GLUT3 promotes glucose uptake and the proliferation of mesenchymal lung tumor cells. (A) Mesenchymal (SW1573) or epithelial
(H727) cells were transfected with control (ctl) siRNA, each of three different siRNAs to decrease GLUT3, or each of two siRNAs to decrease GLUT1, as
indicated. One hundred forty-four hours later, live cells were counted by trypan blue exclusion (n = 3 or 4). (B) 2-Deoxy-D-[3H]glucose (DOG) incorporation
was measured in SW1573 cells stably expressing ctl shRNAs or shRNAs targeting GLUT3 (in pSicoR or pLKO.1 vectors, see ‘Methods’). Data show
means ± s.d. (n = 3) of glucose uptake (nmol) measured for 10 min, normalized to protein concentration. (C) SW1573 cells stably expressing ctl shRNAs
or shRNAs targeting GLUT3 (in pSicoR or pLKO.1 vectors) were prepared in soft agar for anchorage-independent growth. Three weeks later, the number
of colonies was determined (n = 6). (D) H727 cells, either parental or stably expressing a control (ctl) plasmid or a GLUT3 cDNA, were cultured in high
or low glucose (glc) concentrations for 4 days, after which live cells were counted by trypan blue exclusion (n = 7).
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B
OS - GLUT3
Figure 6 GLUT3 expression correlates with poor overall survival and EMT in human NSCLC. (A) Kaplan-Meier plot representing overall
survival comparing tumor samples with high or low GLUT3 levels. The numbers of living patients are indicated for each year and group. HR hazard
ratio, OS overall survival. (B) The correlations between each of GLUT3 (SLC2A3), E-cadherin (CDH1), SNAIL (SNAI1), SLUG (SNAI2), Twist (TWIST1),
vimentin (VIM), ZEB1, and ZEB2 were assessed from the pooled dataset. Spearman correlation coefficients (indicated numbers) were calculated
between the expression values of each pair. 0.1 < P < 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.
[29,31]. In contrast, ZEB1 binding to R-SMADs and the
acetyltransferases p300 and P/CAF promotes target gene
transcription [30,32]. Likewise, in our study, we have
demonstrated that ZEB1 can interact with each of CtBP
and p300 in mesenchymal lung tumor cells and that it is
a direct activator of SLC2A3 gene transcription. Of note,
ZEB1 expression was higher in the mesenchymal,
GLUT3-expressing cell lines compared to the epithelial
cell lines, and ZEB1 was induced prior to GLUT3 in response to TGF-β (see Figures 2A and 4A, and Additional
Table 1 Univariate and multivariate analyses of overall
survival in NSCLC
HR
Lower 0.95 Upper 0.95
P value
(Wald)
Univariate
1.047
1.432
1.14E − 02
SLC2A3 (continuous)
1.224
Stage (II vs. I)
1.590
1.070
2.363
2.19E − 02
Histology LCC vs. AD 1.131
0.458
2.792
7.89E − 01
Histology SCC vs. AD 1.624
1.150
2.293
5.91E − 03
Gender (F vs. M)
0.610
0.429
0.868
6.06E − 03
Age (>62 vs. ≤62)
1.906
1.385
2.622
7.47E − 05
SLC2A3 (continuous)
1.194
1.016
1.404
3.16E − 02
Stage (II vs. I)
1.432
0.945
2.170
9.04E − 02
Histology LCC vs. AD 1.006
0.406
2.495
9.90E − 01
Histology SCC vs. AD 1.552
1.071
2.248
2.03E − 02
Gender (F vs. M)
0.702
0.484
1.020
6.33E − 02
Age (>62 vs. ≤62)
1.829
1.323
2.528
2.56E − 04
Multivariate
AD adenocarcinoma, F female, HR hazard ratio, LCC large cell carcinoma, M male,
SCC squamous cell carcinoma.
files 1 and 4). This is in agreement with a previous
finding reporting that the SLC2A3 gene was one of the
top 50 genes correlating positively with ZEB1 gene expression in a panel of human lung tumor cell lines
[37]. In the future, it will be important to decipher all
of the genes that are directly targeted—either repressed
or induced—by ZEB1 in lung cancer and to determine
if this includes additional genes that regulate tumor cell
metabolism.
The increased GLUT3 expression observed in tumor
cells upon EMT may have important implications for
cancer treatment. Indeed, not only is EMT well recognized as a crucial process that increases invasive capacities of tumor cells and promotes the acquisition of stem
cell characteristics [38], it may occur earlier in tumor development than previously anticipated. For example, in a
K-rasLSL-G12D/+; p53Flox/Flox mouse model of PDAC,
EMT and invasiveness precede the detection of tumors
in situ [39]. Interestingly, a molecular classification of
human PDAC was performed recently, where three subtypes were defined: classical, exocrine-like, and quasimesenchymal (which has the worst prognosis of all
three) [40]; SLC2A3 was one of a 20-gene signature of
the quasi-mesenchymal subtype. This observation, together with our findings of a connection between
GLUT3 and EMT in human liver cells from HCC, indicates that the regulation of GLUT3 by EMT extends beyond lung cancer.
Conclusions
By uncovering direct regulation of GLUT3 by the transcription factor ZEB1, our study provides evidence for a
tight association between two central characteristics of
Masin et al. Cancer & Metabolism 2014, 2:11
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carcinoma development: EMT and glucose metabolism.
Because GLUT3 expression is mainly restricted to the
brain of healthy individuals, the future development of
small molecule compounds that selectively block
GLUT3 and that do not cross the blood-brain barrier
may become a viable strategy to treat patients with
NSCLC and possibly other cancer types.
Additional files
Additional file 1: Figure S1. GLUT1, GLUT4, GLUT12, and ZEB1
expression in mesenchymal and epithelial lung tumor cells. The indicated
cell lines were lysed for RNA preparation followed by reverse transcription.
The cDNA was amplified by real-time PCR using probes specific for the
indicated genes or internal controls. Data show means ± s.d. (n = 3) of
mRNA expression relative to the cell line expressing the least amount of the
same gene (set to 1). When there was no specific amplification after
40 cycles of PCR, the samples were called not determined (N.D.).
Additional file 2: Figure S2. hZEB1 and hGLUT3 induction upon
mSNAIL overexpression. H727 or H2009 cell populations stably expressing
a control plasmid (ctl) or mouse (m) SNAIL were lysed for RNA preparation
followed by reverse transcription. The cDNA was amplified by real-time PCR
using probes specific for human ZEB1, GLUT3, or an internal control. Data
show means ± s.d. (n = 3) of mRNA expression, represented as fold
induction (mSNAIL/ctl) for each cell line. The arrows and numbers indicate
the fold induction between samples.
Additional file 3: Figure S3. ZEB1 interacts with CtBP and p300 in
mesenchymal lung tumor cells. Protein extracts from the indicated cell
lines were used for ZEB1 or control (IgG) immunoprecipitation, followed
by Western blot using the indicated antibodies. The arrowheads indicate
the position of each protein. XT, cell extract; ø, empty lane.
Additional file 4: Figure S4. ZEB1 is induced rapidly upon TGF-β
stimulation. H2122 cells were stimulated with 10 ng/ml TGF-β for the
indicated time points, after which the cells were lysed for RNA preparation
followed by reverse transcription. The cDNA was amplified by real-time PCR
using probes specific for ZEB1, SNAIL, or an internal control. Data show
means ± s.d. (n = 4) of mRNA expression ratios between TGF-β treated and
non-treated conditions.
Additional file 5: Figure S5. Efficiency of siRNA or shRNA knockdown.
(A) Efficiency of GLUT3 or GLUT1 siRNA knockdown. SW1573 cells were
transfected with one of three different siRNAs to target GLUT3, two to
target GLUT1, or a control (ctl) siRNA, or were left untransfected (–).
Seventy-two hours later, the cells were lysed for RNA preparation
followed by reverse transcription. The cDNA was amplified by real-time
PCR using probes specific for the indicated genes or GAPDH as an
internal control. Data show the percentage of mRNA expression relative
to the non-transfected condition (set to 100%). (B) Efficiency of GLUT3
siRNA knockdown. SW1573 cells were transfected with one of three
different siRNAs to target GLUT3, a control (ctl) siRNA, or were left
untransfected (–). Seventy-two hours later, the cells were lysed with RIPA
buffer to prepare protein extracts and to analyze the expression of GLUT3
by Western blot. (C) GLUT3 or GLUT1 knockdown does not affect the
number of dead cells. SW1573 cells were transfected with control (ctl)
siRNA, each of three different siRNAs to decrease GLUT3, or each of two
siRNAs to decrease GLUT1, as indicated. One hundred forty-four hours
later, dead cells were counted by trypan blue exclusion (n = 3). (D) Efficiency
of GLUT3 shRNA stable knockdown. SW1573 cells stably expressing ctl or
GLUT3 shRNAs (in the indicated vectors) were lysed (left panel) with RIPA
buffer to prepare protein extracts and to analyze the expression of GLUT3
by Western blot, or (right panel) for RNA preparation followed by reverse
transcription. The cDNA was amplified by real-time PCR using probes
specific for GLUT3 or GAPDH as an internal control. Data show the
percentage of GLUT3 mRNA expression relative to the control (ctl) shRNA
expressing cells (set to 100%). *n.s., non-specific.
Additional file 6: Figure S6. GLUT3 knockdown leads to diminished
vimentin expression. SW1573 cells stably expressing a control (ctl) or
Page 13 of 14
GLUT3 shRNA were lysed for RNA preparation followed by reverse
transcription. The cDNA was amplified by real-time PCR using probes
specific for the indicated genes or internal controls. Data show means ± s.d.
(n = 3) of mRNA expression relative to the condition with the least amount
of mRNA for each gene (set to 1).
Additional file 7: Figure S7. Generation of H727 cells stably expressing
GLUT3. (A) Stable expression of GLUT3. (Left) Parental H727 cells or H727
cells stably expressing a control (ctl) plasmid or a GLUT3 cDNA were
lysed with RIPA buffer to prepare protein extracts and to analyze the
expression of GLUT3 (or β-tubulin as loading control) by Western blot.
(Right) Parental or GLUT3-expressing H727 cells were stained to analyze
the expression of GLUT3 by immunocytochemistry (ICC). Scale bar,
100 μm. (B) Ectopic GLUT3 expression does not affect cell proliferation in
high glucose concentrations. H727 cells, either parental or stably expressing
a control (ctl) plasmid or a GLUT3 cDNA, were cultured in high glucose (glc)
concentrations for 4 days, after which live cells were counted by trypan blue
exclusion (n = 7).
Abbreviations
EMT: Epithelial-mesenchymal transition; HCC: Hepatocellular carcinoma;
NSCLC: Non-small cell lung cancer; PDAC: Pancreatic ductal adenocarcinoma;
SLC2A3: Solute carrier family 2 (facilitated glucose transporter), member 3;
ZEB1: Zinc finger E-box-binding homeobox 1.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MM participated in the design of the study, performed most of the
experiments, and analyzed the data. JV performed several experiments and
analyzed the data. SR did the bioinformatics analyses of the microarray
datasets. SG carried out the immunoprecipitation experiments and analyzed
the data. NS carried out the ZEB1 knockdown experiments and analyzed the
data. PCS participated in the bioinformatics analyses of the ChIP-seq datasets.
BD participated in the bioinformatics analyses of the ChIP-seq datasets. LEF
carried out the initial real-time PCR experiments of the cell line panel and
analyzed the data. JG, DM, and TGO analyzed the data. EM conceived the
study, participated in its design and coordination, analyzed the data, and
wrote the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We thank Anja Irmisch and Ute Koch (ISREC, EPFL) for their help on the
ChIP-PCR and 2-DOG uptake experiments, respectively, and the EPFL SV
Histology Core Facility for histological sectioning. We thank Tyler Jacks
(Massachusetts Institute of Technology) for insightful comments, and Mikael
J. Pittet (Harvard Medical School) and Inder M. Verma (The Salk Institute) for
critical reading of the manuscript. This work was supported by the Swiss
National Science Foundation (PP00P3_133661) and by a ‘Molecular Life
Sciences’ grant from the ISREC Foundation. The funding bodies played no
role in the design, collection, analysis, interpretation of data, writing, or
decision to submit the manuscript for publication.
Author details
1
Swiss Institute for Experimental Cancer Research, School of Life Sciences,
Ecole Polytechnique Fédérale de Lausanne, Lausanne 1015, Switzerland.
2
Bioinformatics Core Facility, Swiss Institute of Bioinformatics, Lausanne 1015,
Switzerland. 3Institute of Bioengineering, School of Life Sciences, Ecole
Polytechnique Fédérale de Lausanne, Lausanne 1015, Switzerland. 4Koch
Institute for Integrative Cancer Research, Massachusetts Institute of
Technology, Cambridge, MA 02139, USA. 5Division of Gastroenterology and
Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne,
Lausanne 1011, Switzerland. 6Huntsman Cancer Institute, University of Utah,
Salt Lake City, UT 84112, USA.
Received: 27 March 2014 Accepted: 11 July 2014
Published: 29 July 2014
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doi:10.1186/2049-3002-2-11
Cite this article as: Masin et al.: GLUT3 is induced during epithelialmesenchymal transition and promotes tumor cell proliferation in non-small
cell lung cancer. Cancer & Metabolism 2014 2:11.